nucleic acid sequencing protocol
For the direct conversion of human fibroblasts into induced neurons (iN), cells were lentivirally transduced to express rtTA and a 2A-peptide-linked transcript coding for the pro-neuronal transcription factors Ngn2 and Ascl1, resulting in transgenic, but silent and expandable, AN fibroblasts. For iN conversion, cells were transferred to conversion media containing doxycycline and small molecular enhancers (also see Ladewig et al., Nat Methods 2012). Under these conditions, fibroblasts underwent marked morphological changes into neuronal cells, with typical neuronal morphologies appearing around week 2 of conversion. RNA was collected for RNA-Seq on various time points during the iN conversion process. To analyze the transcriptome of iNs via RNA-Seq, neuronal RNA needs to be pure, with minimal fibroblast contamination that might confound analysis. As live iNs express PSA-NCAM on the cell surface, we established a FACS-based protocol for purification of neurons using a PSA-NCAM antibody. Pure iN RNA for RNA-Seq was isolated by sorting directly into Trizol-LS reagent. All other fibroblast and iPSC RNA samples were directly collected from adherent mono-cultures.
nucleic acid library construction protocol
TruSeq stranded mRNA (Illumina)
nucleic acid extraction protocol
Trizol-LS and RNeasy mini